In article <05AUG95.17240381.0069 at VM1.MCGILL.CA>, "SEGALL,LAURA,MS" <BK5U000 at MUSICB.MCGILL.CA> says:
>>Hi!
>I'm working in a lab that was recently set up, and we are having a
>very difficult time isolating RNA from cell cultures. We consistently
>get only about half of the expected amounts, and even then, we seem to
>have some DNA contamination since when we load say 5 ug RNA for a
>Northern, we see a much weaker band than we do for controls, so some-
>thing is interfering in our getting the proper concentration reading.
>We now spin the lysed cells for 45 min. rather than 20 min, and there
>seems to be some improvement, but we now think that there is something
>very seriously wrong with our Guanidine Thiocyanate solution (although
>we use 4M guanidine Hydrochloride with Na-citate and 10% Laurylsarcosyl?
>whatever, the detergent).
>We were told that the pH of the solution should be 7, but ours was at
>5.5. We made a new solution, and got the same thing. When we tried to
>fix the pH, we noticed that the citrate isn't really buffering the lut
>solution at all. We've found detergent precipitates in it and were
>forced to heat it to clear it up a bit, although it didn't do much.
>At this point, any help is welcome.
>Thankyou in advance,
>Laurie.
Laurie,
I have been using a modified guanidinium thiocyanate RNA isolation in
my RT-PCR work. This method appears in a manual supplement by Bauer, Warthoe,
Rohde, and Strauss in PCR METHODS & APPLICATIONS, vol4, issue 2, October 1994,
pg S97-S108. This method has allowed my laboratory to isolated high quality
RNA from all of the cell lines we study; Caco-2, BeWo, K562, C6, 1474C, 1230M.
I only spin my cells at 550 g for 5 min. at 4 C after lysing the cells
with NP-40. This procedure still yields high levels of RNA. In fact most of
the time the RNA must be diluted to a more utilizable concentration.
Your pH should be 7. I had the same problem at first. I found that
if you heat the stock Sarkosyl to 65 C as well as warm the GTC mixed with
the Na-citrate, and water & then mix together the precip. is minimized.
Once in solution I like to filter & then store at room temp. without percip.
at all.
I saw that some one suggested buffering your phenol before use. I
do this by mixing molten mole. bio. grade phenol with an equal amount of 2 M
Na-acetate (pH 5.0) at least 2 days before use and refrigerate. There was
a noticable difference in the yield of RNA from an isolation once I began
using this acid phenol solution.
To remove DNA contamination use an enzyme. Get some RNase-FREE DNase,
it works wonders and is easily removed by an additional phenol/chloroform
extraction. Also this method uses RNasin to protect the precious sample.
Well, I hope I helped some. If you want the procedure but can't find
it e-mail me & I'll forward the one I use to you.
Good luck
John Nelson
-------------------------------------------------------------------------------------------------------------------------------------------------------
(jnelson at bioch)
Dr. E. D. Harris Laboratory
Department of Biochemistry & Biophysics
Texas A&M University
College Station, TX, U. S. A.